Deoxygalactonojirimycin derivatives

ABSTRACT

Novel N-alkyl derivatives of deoxygalactonojirimycin are provided in which said alkyl contains from 3-6 carbon atoms. These novel compounds are useful for selectively inhibiting glycolipid synthesis.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation-in-part of copending application Ser.No. 08/061,645, filed May 13, 1993 now U.S. Pat. No. 5,399,567.

BACKGROUND OF THE INVENTION

This invention relates to novel N-alkyl derivatives ofdeoxygalactonojirimycin (DGJ) in which said alkyl groups contain from3-6 carbon atoms. These novel compounds are useful for selectivelyinhibiting glycolipid synthesis.

In applicants' copending application Ser. No. 08/061,645, filed May 13,1993, now U.S. Pat. No. 5,399,567 certain N-alkyl derivatives ofdeoxynojirimycin (DNJ) are disclosed as effective inhibitors ofglycolipid biosynthesis. N-alkylated derivatives of DNJ were alsopreviously known to be inhibitors of the N-linked oligosaccharideprocessing enzymes, α-glucosidase I and II. Saunier et al., J. Biol.Chem. 257, 14155-14166 (1982); Elbein, Ann. Rev. Biochem. 56, 497-534(1987). As glucose analogues, they also have potential to inhibitglucosyltransferases. Newbrun et al., Arch. Oral Biol. 28, 516-536(1983); Wang et al., Tetrahedron Lett. 34, 403-406 (1993). Theirinhibitory activity against the glucosidases has led to the developmentof these compounds as antihyperglycemic agents and antiviral agents.See, e.g., PCT Int'l. Appln. WO 87/03903 and U.S. Pat. Nos.: 4,065,562;4,182,767; 4,533,668; 4,639,436; 4,849,430; 5,011,829; and 5,030,638.

BRIEF DESCRIPTION OF THE INVENTION

In accordance with the present invention, novel N-alkyl derivatives ofdeoxygalactonojirimycin (DGJ) are provided in which said alkyl containsfrom 3-6 carbon atoms and preferably from 4-6 carbon atoms. These novelcompounds are useful for selectively inhibiting glycolipid synthesis.The length of the N-alkyl chain has been found to be important to saidinhibitory activity since the non-alkylated DGJ and the N-methyl andN-ethyl derivatives of DGJ were each found to be inactive for suchinhibition. The N-propyl derivative of DGJ was partially active. Thus, aminimum alkyl chain length of 3 carbon atoms has been found to beessential for efficacy.

The biosynthesis of glycolipids in cells capable of producingglycolipids can be selectively inhibited by treating said cells with aglycolipid inhibitory effective amount of an N-alkyl derivative of DGJof the present invention. These inhibitory compounds can be used atconcentrations of about 10-fold less than the effective antiviralconcentrations of the corresponding N-alkyl derivatives of DNJ.Illustratively, the N-butyl DGJ is inhibitory of glycolipid biosynthesisat relatively low concentration of about 50 μM compared to the 0.5 mMlevel of concentration of N-butyl DNJ in cell culture systems forα-glucosidase I inhibition (Karlsson et al., J. Biol. Chem. 268, 570-576(1993).

The active N-alkyl derivatives of DGJ have a significant advantagesince, unlike the previously described N-alkyl derivatives of DNJ, theyselectively inhibit biosynthesis of glycolipids without effect either onthe maturation of N-linked oligosaccharides or lysosomalglucocerebrosidase. For example, in contrast to N-butyl DNJ, the N-butylDGJ of the present invention surprisingly does not inhibit theprocessing α-glucosidases I and II or lysosomal β-glucocerobrosidase.Likewise, the only prior reported experimental evidence usingdeoxygalactonojirimycin indicates that N-alkylation(N-heptyldeoxygalactonojirimycin) provides a modest increase in theaffinity towards certain β-glucosidases [Legler & Pohl, Carb. Res. 155,119 (1986)]. The inhibitory results described herein for the novelN-alkylated deoxygalactonojirimycin analogues in which the alkylcontains from 3 to 6 carbon atoms were unexpected in view of thecorresponding activity of related iminosugar compounds.

Further uniqueness of the present invention is seen by the finding thatthe exemplary N-butyl and N-hexyl derivatives of DGJ completelyprevented glycolipid biosynthesis, whereas the N-butyl derivatives ofmannose, fucose and N-acetylglucosamine were without effect onglycolipid biosynthesis.

The inhibitory effect of these compounds on the biosynthesis ofglycolipids is illustrated herein in the myeloid cell line HL-60 and inthe lymphoid cell line H9. These are well-known, widely distributed andreadily available human cell lines. For example, HL-60 cells arepromyelocitic cells described by Collins et al., Nature 270, 347-349(1977). They are also readily available from the American Type CultureCollection, Rockville, Md, U.S.A., under accession number ATCC CCL 240.H9 cells are of lymphoid origin described by Gallo and Popovic, Science224, 497-500 (1984). They are also readily available from the samedepository under accession number ATCC HTB 176.

The inhibition of glycolipid biosynthesis by these N-alkyl derivativesof DGJ is further demonstrated herein by the reduction of the binding ofcholera toxin to the illustrative cell line H9 when cultured in thepresence on N-butyl DGJ. These compounds thus are also useful asanti-microbial agents by inhibiting the and bacterial toxins asillustrated hereinafter in Tables 1 and 2, respectively.

The inhibitory effect upon the biosynthesis of glycolipids is furtherillustrated by the ability of the N-butyl and N-hexyl derivatives of DGJto offset glucoceramide accumulation in a standard, state-of-the-art invitro model of Gaucher's disease. In this model, the murine macrophagecell line WEHI-3B was cultured in the presence of an irreversibleglucocerebrosidase inhibitor, conduritol β epoxide (CBE), to mimic theinherited disorder found in Gaucher's disease. WEHI-3B cells arewell-known, widely distributed and readily available murine macrophagecells. They are described in Cancer Res. 37, 546-550 (1977), and arereadily available from the American Type Culture Collection, Rockville,Md, under accession number ATCC TIB 68. The compounds of the inventionprevent lysosomal glycolipid storage which is useful for the managementof Gaucher's disease and other glycolipid storage disorders asillustrated hereinafter in Table 3. Gaucher's disease is an autosomalrecessive disorder characterized by an impaired ability to degradeglucocerebroside (glucosyl ceramide, Glc-Cer) due to mutations in thegene encoding β-glucocerebrosidase (β-D-glucosyl-N-acylsphingosineglucohydrolase, EC 3.2.1.45). This defect results in the lysosomalaccumulation of Glc-Cer in cells of the macrophage-monocyte system[Barranger and Ginns, in The Metabolic Basis of Inherited Diseases, ed.Scriver et al., pp. 1677-1698, McGraw-Hill, New York, (1989); Beutler,Science 256, 794-799 (1992)]. By slowing the rate of glycolipidsynthesis, the impaired catabolism of Glc-Cer can be offset, therebyleading to the maintenance of a balanced level of Glc-Cer.

The clinical management of Gaucher's disease currently relies uponeither symptomatic treatment of patients or enzyme replacement therapy[Beutler, Proc. Natl. Acad. Sci. USA 90, 5384-5390 (1993)]. In view ofthe prohibitive cost of enzyme replacement therapy and the requirementfor intravenous administration of glucocerebrosidase, an orallyavailable alternative therapy based around substrate deprivationconstitutes a useful alternative. The N-alkyl derivatives of DGJ arewater-soluble sugar analogs and, therefore, orally active. Since theN-alkyl DGJ compounds exhibit fewer complicating enzyme inhibitorycharacteristics than α-and β-glucosidase inhibitors, they alsoconstitute a preferable alternative to the N-alkyl DNJ compounds fortherapeutic management of Gaucher's disease and other glycolipid storagedisorders. The N-alkyl DGJ may also be used in combination withglucocerebrosidase for the treatment of Gaucher's disease.

DETAILED DESCRIPTION OF THE INVENTION

While the specification concludes with claims particularly pointing outand distinctly claiming the subject matter regarded as forming theinvention, it is believed that the invention will be better understoodfrom the following illustrative detailed description of the inventiontaken in conjunction with the accompanying drawings in which:

FIG. 1 shows by one dimensional thin layer chromatography (1D-TLC) acomparison of N-alkylated imino sugars as inhibitors of glycolipidbiosynthesis. 1D-TLC separation was made of HL-60 total cellular lipidslabelled with [¹⁴C]-palmitic acid. Cells were treated with either 0.5 mMN-butyl deoxynojirimycin (NB-DNJ), N-butyl deoxymannojirimycin (NB-DMJ),N-butyl deoxygalactonojirimycin (NB-DGJ) or N-butyl2-acetamido-1,2,5-trideoxy-1,5-imino-D-glucitol (NB-NAG) or untreated(UT). Glycolipid biosynthesis inhibition was detected by the lack ofGlc-Cer, gangliosides and an unknown species (indicated with arrows).Glc-Cer migration was confirmed by inclusion of a [¹⁴C]-Glc-Cer standardon the TLC. The radiolabelled lipid species were visualized byautoradiography.

FIG. 2, in three parts, A, B and C, shows 2D-TLC analysis of HL-60 cellstreated with either NB-DNJ or NB-DGJ. 2D-TLC separation was made oftotal HL-60 lipids labelled with [¹⁴C]-palmitic acid. Cells were treatedwith either 0.5 mM NB-DNJ or NB-DGJ or untreated (UT). Lipids wereassigned as follows (untreated cells, lefthand panel, FIG. 2A): 1,gangliosides; 2, lysophospatidylcholine; 3, ceramide phosphorylcholine;4, ceramide phosphorylethanolamine; 5, phospatidylcholine; 6,phosphatidylinositol; 7, phosphatidylethanolamine; 8,phosphatidylglycerol; 9, diglycosylceramide; 10, monoglycosylceramide;11, cholesterol/fatty acids/neutral lipids; N and N* are unknowns; and 0is the sample origin. Following NB-DNJ and NB-DGJ treatment (middle andrighthand panels, FIGS. 2B and 2C, respectively) species 1(gangliosides); 9 (diglycosylceramide); 10 (monoglycosylceramide) and N*(unknown) were absent. The radiolabelled lipids were visualized byautoradiography.

FIG. 3 shows the dose dependent effects of NB-DNJ and NB-DGJ onglycolipid biosynthesis. 1D-TLC analysis was made of total cellularlipids. HL-60 cells were labelled with [¹⁴C]-palmitic acid in thepresence or absence (UT) of NB-DNJ or NB-DGJ at the indicatedconcentrations (μM). The migration position of [¹⁴C]-Glc-Cer isindicated by arrows. The lipids were visualized by autoradiography.

FIG. 4, in two parts, A and B, shows the effects of increasing DNJ andDGJ N-alkyl chain length on inhibition of glycolipid biosynthesis.1D-TLC analysis was made of total cellular lipids. HL-60 cells weretreated with [¹⁴C]-palmitic acid in the presence or absence (UT) ofeither DNJ, or the N-ethyl, N-methyl, N-propyl, N-butyl and N-hexylderivatives of DNJ (lefthand panel, FIG. 4A) or DGJ, or the N-ethyl,N-methyl, N-propyl, N-butyl and N-hexyl derivatives of DGJ (righthandpanel, FIG. 4B) at 0.5 mM concentration. The migration position of[¹⁴C]-Glc-Cer is indicated with arrows. The lipids were visualized byautoradiography.

FIGS. 5 and 6 show the analysis of NB-DNJ and NB-DGJ in an in vitroGaucher's disease model. Specifically, FIG. 5 shows the 1D-TLC analysisof glycolipids from WEHI-3B cells treated with either NB-DNJ or NB-DGJ,at the indicated concentrations (AM), and visualized by chemicaldetection (see methods hereinafter).

FIG. 6, in eight parts, A through H, shows the transmission electronmicroscopy of WEHI-3B cell lysosomes: FIG. 6A, untreated; FIG. 6B,conduritol β epoxide (CBE) treated; FIG. 6C, CBE and 500 μM NB-DNJ; FIG.6E, CBE and 50 μM NB-DNJ; FIG. 6G, CBE and 5 μM NB-DNJ; FIG. 6D, CBE and500 μM NB-DGJ; FIG. 6F, CBE and 50 μM NB-DGJ; FIG. 6H, CBE and 5 μMNB-DGJ. The scale bar at the lower right hand corner of FIG. 6H isapplicable to all of FIGS. 6A through H and represents 0.1 μM.

FIG. 7 shows the effect of NB-DGJ on N-linked oligosaccharideprocessing. Specifically, it shows Endo H sensitivity of gp120 expressedin Chinese hamster ovary (CHO) cells in the presence or absence (−) ofeither NB-DNJ or NB-DGJ (0.5 mM and 5 mM). The arrows indicate themolecular weight of the untreated gp120 (120 kDa) and post endo Hdigestion (60 kDa). An additional band of low molecular weight(approximately 60 kDa) was present in some lanes and is a non-specificprotein precipitated by the solid phase matrix.

FIG. 8 is a graphical representation that shows, in three parts, A, Band C, the effect of imino sugar analogues on glycolipid andglycoprotein metabolizing enzyme activity. Enzyme activity wasdetermined in the presence of the following test compounds: DNJ, (♦);NB-DNJ, (▪); DGJ, (▴); NB-DGJ, () at concentrations shown (see methodshereinafter). FIG. 8A, UDP-glucose:N-acylsphingosineglucosyltransferase; FIG. 8B, β-glucocerebrosidase; FIG. 8C, processingα-glucosidase. Enzymatic activity is expressed as a percentage ofcontrol reactions that contained no test compound.

FIG. 9 shows the laser desorption mass spectrometry of N-butyldeoxygalactonojirimycin with a molecular weight of 220 (M+H) andobtained in greater than 95% purity.

FIG. 10 shows the ¹H NMR spectrum of N-butyl deoxygalactonojirimycin.

FIG. 11 is a graphical representation of a cholera toxin binding assayand shows on the y-axis the % reduction in cholera toxin binding sitesper cell for H9 cells in which the cholera toxin was fluoresceinconjugated and in which the levels of binding to the cell surfaces ofuntreated (ut) cells and cells treated with N-butyldeoxygalactonojirimycin (NB-DGJ) or, for comparison, N-butyldeoxynojirimycin (NB-DNJ), at various levels shown on the x-axis(mg/ml), were measured by flow cytometry.

In order to further illustrate the invention, the following detailedexamples were carried out although it will be understood that theinvention is not limited to these specific examples or the detailstherein.

EXAMPLES MATERIALS & METHODS

Compounds:

N-Butyldeoxynojirimycin (NB-DNJ) was obtained from Searle/Monsanto (St.Louis, Mo., U.S.A.). Deoxygalactonojirimycin (DGJ), deoxyfuconojirimycin(DFJ), deoxymannojirimycin (DMJ), and2-acetamido-1,2,5-trideoxy-1,5-imino-D-glucitol (NAG), were obtainedfrom Cambridge Research Biochemicals (Northwich, Cheshire, U.K.). DGJ,DFJ, DMJ and NAG were reductively N-alkylated in the presence ofpalladium black under hydrogen using the appropriate aldehyde byconventional procedure as described by Fleet et al., FEBS Lett. 237,128-132 (1988). The reaction mixture was filtered through Celite and thesolvent removed by evaporation under vacuum. The resulting N-alkylatedanalogues were purified by ion-exchange chromatography (DOWEX® AG50-X12,H+ form) in 2M NH₃ (aq) and the solvent removed by evaporation. Thesecompounds were lyophilised and analysed by 1D ¹H NMR at 500 MHz on aVarian Unity 500 spectrophotometer and by matrix assisted laserdesorption (Finnegan). All compounds synthesised were greater than 95%pure. The following are representative examples of the synthesis of theforegoing N-alkylated compounds as used hereinafter.

Example 1

In a representative example of the preparation of the N-butyldeoxygalactonojirimycin, 30 mg (184 μmol) of deoxygalactonojirimycin wasdissolved in 1 ml of 50 mM sodium acetate buffer, pH 5.0, to which 20 mgof palladium black was added. A hydrogen atmosphere was maintained inthe reaction vessel and 100 μl (1.1 mmol) of butyraldehyde wasintroduced. The reaction was stirred for 16 hr. at room temperature (ca.20° C.). The reaction was stopped by filtration through a bed (1 g) ofCelite (30-80 mesh) and the reaction products were separated bychromatography using a column containing 4 ml of packed DOWEX® AG50-X12(H+ form) resin. The N-butyl deoxygalactonojirimycin was eluted from thechromatography column with 2M ammonia. Its molecular mass was 220 (M+H)as determined by laser desorption mass spectrometry and its chemicalstructure was confirmed by 1D ¹H NMR as shown in FIGS. 9 and 10,respectively.

Example 2

The synthesis procedure and compound analysis of Example 1 was repeatedexcept that caproaldehyde was substituted for an equivalent amount ofbutyraldehyde for analogous preparation of N-hexyldeoxygalactonojirimycin. Its molecular mass was 248 (M+H) as determinedby laser desorption mass spectrometry and its chemical structure wasconfirmed by 1D ¹H NMR.

Example 3

The synthesis procedure and compound analysis of Example 1 was repeatedexcept that propanoyl aldehyde was substituted for an equivalent amountof butyraldehyde for analogous preparation of N-propyldeoxygalactonojirimycin. Its molecular mass was 206 (M+H) as determinedby laser desorption mass spectrometry and its chemical structure wasconfirmed by 1D ¹H NMR.

The N-alkylated deoxygalactonojirimycin compounds prepared in theforegoing illustrative Examples 1 to 3 were obtained in overall yieldsof 68-74% based on the starting deoxygalactonojirimycin and were greaterthan 95% pure.

Enzymes and Enzyme Assays:

Porcine liver α-glucosidase I and rat liver α-glucosidase II werepurified to homogeneity and assayed by conventional procedure using a[¹⁴C]-glucose labelled Glc₃Man₉GlcNAc₂ substrate as previously describedby Karlsson et al., J. Biol. Chem. 268, 570-576 (1993).

β-D-Glucosyl-N-acylsphingosine glucohydrolase (glucocerebrosidase) wasisolated from human placenta and purified to homogeneity according topublished standard methods [Furbish et al., Proc. Natl. Acad. Sci. USA74, 3560-3563 (1977); Dale and Beutler, Ibid. 73, 4672-4674 (1976)].Glucocerebrosidase activity was measured by adding enzyme (5-50 μl) to asonicated suspension of buffer (50 μl of 50 mM sodium citrate/sodiumphosphate buffer, pH 5.0) containing glucosyl ceramide (1 mM), TRITON®X-100 non-ionic surfactant (0.25% v/v) and sodium taurodeoxycholate(0.6% v/v) that had been previously dried under nitrogen fromchloroform:methanol (2:1 v/v) solutions. After incubation at 37° C. for15-60 min., the reaction was stopped by the addition of 500 μl ofchloroform:methanol and the phases separated by centrifugation. Theupper phase was washed twice with Folch theoretical lower phase [Folchet al., J. Biol. Chem. 226, 497-509 (1957)] desalted using AG50-X12ion-exchange resin and dried under vacuum. The reaction products wereseparated by high performance anion exchange chromatography (DionexBioLC System) and detected by pulsed amperometry. The amount ofenzyme-released glucose was calculated from peak areas by applyingexperimentally determined response factors for glucose relative to anincluded reference monosaccharide [Butters et al, Biochem. J. 279,189-195 (1991)].

UDP-glucose:N-acylsphingosine glucosyltransferase (EC 2.4.1.80) activitywas measured in rat brain homogenates and mouse macrophage tissuecultured cell (WEHI-3B) homogenates using a method adapted as followsfrom published conventional procedures [Vunnam and Radin, Chem. & Phys.of Lipids 26, 265-278 (1980); Shukla and Radin, Arch. Biochem. Biophys.283, 372-378 (1990)]: Dioleoylphosphatidylcholine and cerebrosideliposomes containing 200 nmol ceramides Type IV (Sigma) were added to areaction mixture (100 μl) composed of 40 mM2-[N-morpholino]ethanesulfonic acid (MES) buffer, pH 6.5, 5 mM MnCl₂,2.5 mM MgCl₂, 1 mM NADH and 8 μM UDP-[¹⁴C]-glucose (318 mCi/mmol,Amersham International, Amersham, U.K.). After incubation at 37° C. for1-2 hr. the reaction was stopped by the addition of EDTA (25 mM) and KCl(50 mM). Radiolabelled glycolipids were extracted with 500 μl ofchloroform:methanol (2:1 v/v) for 10 min. and the phases separated. Thelower phase was washed twice with Folch theoretical upper phase andportions taken for scintillation counting. When imino sugars were testedfor inhibitory activity, these were added at appropriate concentrationsto homogenates and preincubated for 10 min. before sonication withceramide containing liposomes. Control reactions were performed withliposomes containing no ceramide to measure the activity of transfer toendogenous acceptors.

Glycolipid Analysis:

HL-60 cells were cultured by conventional procedure as previouslydescribed by Platt et al., Eur. J. Biochem. 208, 187-193 (1992). HL-60cells at 5×10⁴ cells/ml were cultured in the presence or absence ofimino sugars for 24 hr. For labelling, the two dimensional thin layerchromatography (2D-TLC) conventional method of Butters and Hughes wasfollowed [In Vitro 17, 831-838 (1981)]. Briefly, [¹⁴C]-palmitic acid(56.8 mCi/mmol, ICN/Flow) was added as a sonicated preparation in foetalcalf serum (FCS, Techgen, London, U.K., 0.5 μCi/ml) and the cellscultured for a further 3 days maintaining the imino sugars in themedium. The cells were harvested, washed three times with phosphatebuffered saline (PBS), extracted in 1 ml chloroform:methanol (2:1 v/v)and separated by 1 dimensional TLC, loading equal counts (1D-TLC,chloroform:methanol:water (65:25:4)). For two dimensional separationsthe one dimensional separation was performed as described above, theplate dried overnight under vacuum and separated in the second dimensionusing a solvent of tetrahydrofuran: dimethoxymethane:methanol:water(10:6:4:1). Plates were air dried and exposed to Hyperfilm-MP highperformance autoradiography film (Amersham).

Cell Culture and Metabolic Labelling:

The culture of CHO cells expressing soluble recombinant gp120 (from Dr.P. Stevens, MRC AIDS Directed Programme Reagent Project) and theradiolabelling of these cells was carried out by conventional procedureas described by Karlsson et al., J. Biol. Chem. 268, 570-576 (1993).Briefly, CHO cells were harvested mechanically, washed three times withphosphate buffered saline, 0.1M pH 7.2 (PBS) and resuspended inmethionine- and cysteine-free RPMI-1640 medium (ICN-Flow Laboratories,High Wycombe, Bucks, U.K.) supplemented with 1% dialysed FCS. Cells(10⁷/ml) were preincubated in the presence or absence of NB-DNJ orNB-DGJ for 1 hr prior to the addition of 100 μCi/ml Tran³⁵S-label(ICN-Flow) for 4 hr. The supernatants were collected and concentratedtenfold using a 30 kDa cut-off membrane (Amicon, Danvers, Mass.,U.S.A.).

Immunoprecipitation:

Immunoprecipitations were performed by conventional procedure asdescribed by Karlsson supra. Supernatants were incubated with the mAbABT 1001 monoclonal antibody (American Biotechnologies Inc., Cambridge,Mass., U.S.A.) at 0.5 μg/100 μl of supernatant for 30 min. at roomtemperature followed by sheep anti-mouse IgG1-coated magnetic beads(Dynal Ltd., Wirral, Merseyside, U.K., 1.2×10⁷ beads per sample) for 1hr. at 4° C. The beads were washed three times with 2% TRITON® X-100 inPBS and three times with PBS. Gp120 was eluted in 100 μl reducingSDS-PAGE sample buffer with heating (95° C., 5 min.). Each sample wasdivided into two equal aliquots and 25 μl of dH₂O added to give a finalvolume of 50 μl. To one half of each sample 2 μl of endoglycosidase H(endo H, 1 unit/ml, Boehringer Mannheim Ltd., Lewes, Sussex, U.K.) wasadded and the other half left untreated. Digestion was performed at 37°C. for 18 hr. and terminated by the addition of 50 μl of SDS-PAGEreducing sample buffer (95° C., 5 min.).

Glycopeptide Analysis:

HL-60 and BW5147 cells were cultured in RPMI-1640 and 10% FCS. The cellswere incubated for 30 min. in the presence or absence of 2 mM NB-DNJ orNB-DGJ in reduced glucose RPMI-1640 medium (Flow), supplemented with 1%dialysed FCS. [³H]-mannose (16.5 Ci/mmol, Amersham) was added at 200μCi/ml and the cells cultured for a further 3 hr. Washed cell pelletswere resuspended in 50 mM TrisHCl buffer, pH 7.5, containing 10 mM CaCl₂and 0.02% sodium azide and heated at 100° C. for 5 min. After cooling,Pronase® enzyme was added to 0.04% (w/v final concentration) andincubated for 96 hr. at 37° C. under toluene with aliquots of Pronase®added at each 24 hr. period. The digestion was stopped by boiling for 5min., and glycopeptides recovered by centrifugation at 13000 g for 10min. Samples were fractionated by Con A-SEPHAROSE® chromatographyaccording to conventional procedure of Foddy et al., Biochem. J. 233,697-706 (1986).

In Vitro Gaucher's Disease Model:

The in vitro Gaucher's disease model was prepared as follows: WEHI-3Bcells (American Type Culture Collection, Rockville, Md., U.S.A.) weremaintained in logarithmic phase growth for 14 days in RPMI-1640 medium,10% FCS, in the presence or absence of 50 μM conduritol β epoxide (CBE,Toronto Research Chemicals, Downsview, Canada) with or without NB-DNJ orNB-DGJ. Cells were passaged every 3 days and compound concentrationsmaintained throughout. Equal cell numbers (5×10⁶) were harvested,extracted in 1 ml chloroform: methanol (2:1 v/v) overnight at 4° C., theextracts centrifuged, the chloroform:methanol extract retained and thepellet re-extracted as above for 2 hr. at room temperature. Pooledextracts were dried under nitrogen, re-dissolved in 10 μlchloroform:methanol (2:1 v/v) and separated by 1D-TLC inchloroform:methanol:water (10:6:4:1). Plates were air dried andvisualized using α-naphthol (1% w/v in methanol) followed by 50% (v/v)sulphuric acid.

Transmission Electron Microscopy:

Cells for electron microscopy were harvested (1×10⁷ cells pertreatment), washed three times in serum free RPMI-1640 medium and fixedin medium containing 2% glutaraldehyde (v/v) and 20 mM Hepes (v/v) onice for 2 hr. Cells were washed in 0.1 M cacodylate buffer containing 20mM calcium chloride (w/v). Cells were post-fixed with 1% osmiumtetroxide in 25 mM cacodylate buffer (w/v) containing 1.5% potassiumferrocyanide (w/v) for 2 hr. on ice. Samples were dehydrated through anethanol series, transferred to propylene oxide and embedded in Embed 800(Electron Microscopy Sciences, PA, U.S.A.). The sections were stainedwith uranyl acetate/lead citrate and observed with a Hitachi 600microscope at 75 kv.

Analysis of Cholera Toxin Binding to the H9 Human Lymphoid Cell LineFollowing Three Days Treatment with NB-DNJ or NB-DGJ:

Methods: Cells were maintained in logarithmic phase growth in RPMI-1640medium. Cholera toxin B chain (Sigma) was conjugated to fluoresceinisothiocyanate (Sigma) and flow cytometric analysis was carried out byconventional procedure as described by Platt et al., Eur. J. Biochem.208, 187-193 (1992). Analysis was performed on a FACScan Cytometer(Becton Dickinson, Sunnyvale, Calif., USA). Data on viable cells werecollected on a four decade log₁₀ scale of increasing fluorescenceintensity. The data are presented as percent reduction in cholera toxinbindings sites per cell on the y-axis against compound concentration onthe x-axis. The specificity of cholera toxin : cell surface binding wasestablished by inhibiting this interaction with a one hundred fold molarexcess of GMI derived oligosaccharide,GalβGalNAcβ4(NeuAcα3)Galβ4Glcβ3Cer.

RESULTS

Comparison of N-alkylated Imino Sugars as Inhibitors of GlycolipidBiosynthesis:

The glucose analogue, NB-DNJ and four pyranose analogues (NB-DMJ,mannose analogue; NB-DFJ, fucose analogue; NB-DGJ, galactose analogue;and NB-NAG, N-acetylglucosamine analogue) were assessed by the abovemethods for their capacities to inhibit the metabolic incorporation ofradiolabelled palmitate into glycolipids in HL-60 cells at a 500 μMcompound concentration using 1D-TLC analysis (FIG. 1). In addition toNB-DNJ, the only analogue which specifically inhibited glycolipidbiosynthesis was NB-DGJ. All other analogues were without effect. BothNB-DNJ and NB-DGJ inhibited the biosynthesis of Glc-Cer, gangliosidesand an unknown lipid species in agreement with the previous observationswith NB-DNJ described in copending application Ser. No. 08/061,645. Toconfirm that NB-DNJ and NB-DGJ had comparable effects on the completespectrum of glycolipids in this cell line, 2D-TLC was performed toresolve further the individual glycolipid species (FIG. 2). A totaldepletion of glycolipid species was achieved with both 500 μM NB-DNJ andNB-DGJ. Specifically, gangliosides, the unknown lipid (N*) and both themono and dihexaside species were absent following treatment with eithercompound. Phospholipid composition and relative abundance werecomparable, irrespective of treatment, consistent with the previousobservations in copending application Ser. No. 08/061,645 thatN-alkylated imino sugars have no effect on sphingomyelin or phospholipidbiosynthesis. When the two analogues were compared at a range ofconcentrations by 1D-TLC (FIG. 3) both analogues exhibited completeglycolipid inhibition between 50 μM and 500 μM concentrations, althoughpartial inhibition occurred with both compounds at concentrations as lowas 0.5-5 μM. Both analogues were non-cytotoxic in the dose range tested.

Effects of Increasing DNJ and DGJ N-alkyl Chain Length on Inhibition ofGlycolipid Biosynthesis:

A series of N-alkylated DNJ and DGJ derivatives were compared for theirabilities to inhibit glycolipid biosynthesis (FIGS. 4A and 4B,respectively) by 1D-TLC. The non-alkylated imino sugars and the N-methylDNJ, N-methyl DGJ and N-ethyl DGJ had no effect on glycolipidbiosynthesis. The N-propyl analogues of both parent compounds showedpartial inhibitory activity, whereas the N-butyl and N-hexyl derivativesof DNJ and DGJ completely inhibited glycolipid biosynthesis, asdetermined by the loss of detectable Glc-Cer. These data were thereforein agreement with the data from the previous application Ser. No.08/061,645 (where the N-methyl derivative was compared with N-butyl andN-hexyl DNJ). There is a minimal N-alkyl chain length requirement toachieve full inhibition of glycolipid biosynthesis, with butyl and hexylbeing optimal.

Analysis of NB-DGJ in an in Vitro Gaucher's Disease Model:

The WEHI-3B murine macrophage cell line can be induced to resembleGaucher's cells by treatment with the irreversible glucocerebrosidaseinhibitor CBE. NB-DNJ and NB-DGJ were compared in their ability toprevent the accumulation of Glc-Cer in this system (FIG. 5). Bothanalogues prevented CBE induced glycolipid storage in the 5-50 μM doserange. These data therefore demonstrate that NB-DGJ is as effective asNB-DNJ in preventing glycolipid storage in this in vitro Gaucher'sdisease model. The status of the lysosomes from cells treated witheither NB-DNJ or NB-DGJ was assessed by transmission electron microscopy(FIG. 6). It was found that both analogues prevented the glycolipidaccumulation observed in the lysosomes of cells treated with CBE.

Specificity of NB-DGJ for the Glycolipid Biosynthetic Pathway:

The CHO cell line is unique in that it lacks significant levels of theGolgi endomannosidase which acts to circumvent α-glucosidase I and IIinhibition [Karlsson et al., J. Biol. Chem. 268, 570-576 (1993);Hiraizumi et al., J. Biol. Chem. 268, 9927-9935 (1993)]. As aconsequence, it offers an unambiguous cellular system in which to testα-glucosidase inhibition. NB-DNJ was previously tested in this cell lineexpressing recombinant gp120 and it was found that it results in themaintenance of glucosylated high mannose oligosaccharides on gp120 whichare fully sensitive to endo H [Karlsson et al., supra].

Analysis of the N-linked oligosaccharides of gp120 expressed in CHOcells was performed in the presence or absence of NB-DNJ or NB-DGJ (FIG.7). Treatment of CHO cells with 0.5 mM or 5 mM NB-DNJ resulted in fullyendo H sensitive gp120 N-linked glycans in contrast to the untreatedgp120 which was partially sensitive to endo H. This partial sensitivityof untreated gp120 to endo H is because gp120 carries approximatelyfifty percent high mannose N-linked oligosaccharides per molecule[Mizuochi et al., Biochem. J. 254, 599-603 (1988); Mizuochi et al.,Biomed. Chrom. 2, 260-270 (1988)]. However, when the galactose analogue,NB-DGJ, was tested in this system, at 0.5 mM and 5 mM concentrations,gp120 remained partially sensitive to endo H and was indistinguishablefrom the untreated gp120 molecules. This suggested that the galactoseanalogue was not acting as an inhibitor of α-glucosidases I and II.

To examine the effect on endogenous glycoprotein synthesis,radiolabelled glycopeptides were isolated from treated HL-60 and murineBW5147 cells and analysed for their affinity for Con A-SEPHAROSE®. Thisprocedure efficiently resolves tetra- and tri-antennary complexN-glycans from bi-antennary and high mannose/hybrid N-glycans [Foddy etal., Biochem. J. 233, 697-706 (1986)]. Addition of NB-DNJ changes theaffinity of glycopeptides eluting from the Con A-SEPHAROSE® column(Table 4) as a result of processing glucosidase inhibition. Thus theproportion of unbound glycans (tetra- and tri-antennary species)decreases, and a corresponding increase is found in the proportion ofhigh mannose/hybrid glycans that are tightly bound to Con A-SEPHAROSE®and eluted with 500 mM methylmannoside. Similar gross changes inglycopeptide composition following treatment with α-glucosidaseinhibitors are well established [Moore and Spiro, J. Biol. Chem. 265,13104-13112 (1990)]. The galactose analogue, NB-DGJ, showed an unchangedglycopeptide profile by Con A-SEPHAROSE® chromatography (Table 4). Toconfirm these data, glucosidase inhibition was measured directly invitro using a mixture of purified α-glucosidases I and II (FIG. 8).Whereas NB-DNJ inhibited glucosidase I and II with an IC₅₀ of 0.36 μM,NB-DGJ was only weakly inhibitory (IC₅₀ of 2.13 mM, Table 5). These dataprovide substantial evidence that in both in vitro α-glucosidase assaysand in intact cellular system assays NB-DGJ does not inhibit N-linkedoligosaccharide processing.

DNJ and its N-alkylated derivatives are inhibitors of the purifiedlysosomal glucocerebrosidase enzyme required for the cleavage of Glc-Certo glucose and ceramide [Osiecki-Newman et al., Biochim. Biophys. Acta.915, 87-100 (1987)]. In recent tests with the in vitro Gaucher's diseasemodel in co-pending application Ser. No. 08/061,645, it was observedthat WEHI-3B cells incubated in the absence of CBE but in the presenceof NB-DNJ accumulated Glc-Cer. It was therefore apparent that theN-butyl derivative of DNJ was also acting as an inhibitor ofglucocerebrosidase in a cellular environment. The inhibitory activity ofNB-DNJ and NB-DGJ was therefore directly measured to investigatequantitatively their capacities to inhibit human placentalglucocerebrosidase (Table 5). NB-DNJ provided moderate inhibition ofcatalysis with an IC₅₀ of 0.52 mM while NB-DGJ did not inhibit enzymeactivity even at the highest concentration tested (1 mM). In terms ofpercent enzyme inhibition achieved with the two analogues, 1 mM NB-DNJresulted in 90% inhibition while 1 mM NB-DGJ was non-inhibitory (FIG.8), thereby further confirming the advantageous and unexpected selectiveinhibitory activity of NB-DGJ compared to that of NB-DNJ.

Inhibition of UDP-glucose:N-acylsphingosine Glucosyltransferase:

The determination of transferase activity using rat brain or mousemacrophage tissue cultured cells followed saturation kinetics for bothexogenously added ceramide acceptor and UDP-glucose donor. Under theseconditions both N-butylated DNJ and DGJ were moderate inhibitors ofglucose transfer, (IC₅₀ 2.95 mM and 60.88 mM, respectively, Table 5)whereas their unmodified parent homologues were not inhibitory at thehighest concentration tested 6.1 and 5.0 mM, respectively, FIG. 8).

Analysis of Cholera Toxin Binding to the H9 Human Limphoid Cell LineTreated with NB-DGJ:

The activity of the representative N-butyl deoxygalactonojirimycin(NB-DGJ) for inhibiting the surface expression of glycolipid receptorsfor bacteria and bacterial toxins was illustrated by subjecting H9 cellsto cholera toxin binding sites in the presence of varying concentrationsof the NB-DGJ. As a specific probe, advantage was taken of the GM1binding specificity of the cholera toxin B chain [van Heyningen, Nature249, 415-417 (1974); Karlsson, Ann. Rev. Biochem. 58, 309-350 (1989)].The binding of cholera toxin to H9 cells cultured in the presence ofNB-DGJ was reduced by approximately 70% (FIG. 11). This was consistentwith the loss of GM1 from the cell surface and provided further evidencefor the inhibition of glycolipid biosynthesis by NB-DGJ, even though bycomparison it was less than the approximately 90% reduction (FIG. 11)obtained with the N-butyl deoxynojirimycin (NB-DNJ). These resultsdemonstrate that the imino sugar derivatives have use as anti-microbialagents by inhibiting the surface expression of glycolipid receptors forbacteria and bacterial toxins as shown in Tables 1 and 2, respectively.

TABLE 1 GLYCOSPHINGOLIPID RECEPTORS FOR BACTERIAL CELLS MicroorganismTarget Issue Presumed Specificity E. coli Urinary Galα4Galβ E. coliUrinary GlcNAcβ Propionibacterium Skin/Intestine Galβ4Glcβ Severalbacteria Diverse Galβ4Glcβ Streptococcus pneumoniae RespiratoryGlcNAcβ3Gal E. coli CFA/I Intestine NeuAcα8 E. coli Urinary NeuAcα3GalE. coli Intestine NeuGcα3Galβ4GlcβCer GalNAcβ4(NeuAcα3)- Galβ4GlcβCerStaphylococcus Urinary Galβ4GlcNAc saprophyticus Actinomyces naeslundiMouth Galβ, GalNAcβ, Galβ3GalNAcβ, GalNacβ3Galβ Pseudomonas RespiratoryGalNAcβ4Gal Neisseria gonorrhoeae Genital Galβ4Glcβ NeuAcα3Galβ4GlcNAc

TABLE 2 GLYCOSPHINGOLIPID RECEPTORS FOR BACTERIAL TOXINS PresumedReceptor Microorganism Toxin Target Tissue Sequence Vibrio choleraeCholera toxin Small Intestine Galβ3GalNAcβ4- (NeuAcα3)Gal- β4GlcβCer E.coli Heat-labile Intestine Galβ3GalNAcβ4- toxin (NeuAcα3)Gal- β4GlcβCerClostridium Tetanus toxin Nerve Galβ3GalNAcβ4- tetani (NeuAcαβ8Neu-Acα3)Galβ4Glc- βCer Clostridium Botulinum Nerve NeuAcα8NeuAcα- botulinumtoxin A and E Membrane 3Galβ3GalNAcβ- 4(NeuAcα8Neu- Acα3)Galβ4Glc- βCerClostridium Botulinum Nerve NeuAcα3Galβ3- botulinum toxin B, C MembraneGalNAβ4(Neu- and F Acα8NeuAcα3)- Galβ4GlcβCer Clostridium BotulinumNerve GalβCer botulinum toxin B Membrane Clostridium Delta toxin Celllytic GalNAcβ4- perfringens (NeuAcα3)Galβ- 4GlcβCer Clostridium Toxin ALarge Galα3GalβGlc- difficile Intestine NAcβ3Galβ4- GlcβCer ShigellaShiga toxin Large Galα4GalβCer dysenteriae Intestine Galα4Galβ4Glc-GlcNAcβ4Glc- NAc E. coli Vero toxin or Intestine Galα4Galβ4- Shiga-likeGlcβCer toxin

TABLE 3 HERIDITARY GLYCOLIPID STORAGE DISORDERS Disease LipidAccumulation Enzyme Defect Gaucher's GlucocerebrosideGlucocerebroside-β- glucosidase Ceramide Lactoside Ceramide LactosideCeramidelactoside-β- Lipidosis galactosidase Fabry's CeramideTrihexoside Ceramidetrihexoside-α- galactosidase Tay-Sach's GangliosideGM2 Hexosaminidase A Sandhoff's Globoside and GM2 Hexosaminidase A and BGeneral Ganglioside GM1 β-Galactosidase Gangliosidosis FucosidosisH-isoantigen α-Fucosidase Krabbe's GalactocerebrosideGalactocerebroside-β- galactosidase Metrachromatic Sulfatide SulfatidaseLeukodystrophy

TABLE 4 EFFECT OF IMINO SUGAR ANALOGUES ON OLIGOSACCHARIDE BIOSYNTHESISTotal ³H- Tetra- & Oligo- mannose Tri- Bi- mannose recovered Cell lineTreatment antennary antennary & hybrid (cpm) HL-60 untreated 28.3 18.753.0 666918 NB-DNJ 19.5 20.0 60.5 913095 NB-DGJ 28.1 17.7 54.2 844322BW5147 untreated 46.1 5.6 48.3 476527 NB-DNJ 26.8 4.9 68.3 686026 NB-DGJ40.4 7.2 52.4 706873

Cells were radiolabelled for 4 hr. with [³H]-mannose in the presence orabsence of compounds as shown above. Washed cells were exhaustivelydigested with Pronase® enzyme and resultant glycopeptides fractionatedby Con A-SEPHAROSE® chromatography as described hereinbefore. Thepercentage of radiolabelled glycopeptides that were non-bound (complextetra- and tri-antennary N-glycans), eluted with 10 mM methylglucoside(complex bi-antennary N-glycans), or further eluted with 500 mMmethylmannoside (oligomannose and hybrid N-glycans) were calculated fromestimations of radioactivity recovered from pooled eluates.

TABLE 5 CONCENTRATIONS OF IMINO SUGAR ANALOGUES REQUIRED FOR THEINHIBITION OF GLYCOLIPID AND GLYCOPROTEIN METABOLISING ENZYMES CompoundIC₅₀ values Enzyme DNJ NB-DNJ DGJ NB-DGJ UDP-glucose:N- —† 2.95 mM —†60.88 mM acylsphingosine glucosyltransferase β-glucocerebrosidase 2.43mM 0.52 mM —* —* α-glucosidase I and II nd 0.36 μM nd  2.13 mM *notinhibitory at 1 mM concentrations of compound. † not inhibitory at thehighest concentrations tested (see FIG. 8) nd not determined Enzymeswere assayed according to procedure described hereinbefore usingconcentrations of analogues shown in FIG. 8. The data from FIG. 8 wereplotted on a logarithmic scale for accurate estimations of IC₅₀ values,shown above.

In addition to their use as inhibitors of glycolipid biosynthesis incells, the inhibitory agents described herein also can be used foradministration to patients afflicted with glycolipid storage defects byconventional means, preferably in formulations with pharmaceuticallyacceptable diluents and carriers. These agents can be used in the freeamine form or in their salt form. Pharmaceutically acceptable saltderivatives are illustrated, for example, by the HCl salt. The amount ofthe active agent to be administered must be an effective amount, thatis, an amount which is medically beneficial but does not present toxiceffects which overweigh the advantages which accompany its use. It wouldbe expected that the adult human daily dosage would normally range fromabout one to about 100 milligrams of the active compound. The preferableroute of administration is orally in the form of capsules, tablets,syrups, elixirs and the like, although parenteral administration alsocan be used. Suitable formulations of the active compound inpharmaceutically acceptable diluents and carriers in therapeutic dosageform can be prepared by reference to general texts in the field such as,for example, Remington's Pharmaceutical Sciences, Ed. Arthur Osol, 16thed., 1980, Mack Publishing Co., Easton, Pa., U.S.A.

Various other examples will be apparent to the person skilled in the artafter reading the present disclosure without departing from the spiritand scope of the invention. It is intended that all such other examplesbe included within the scope of the appended claims.

What is claimed:
 1. N-alkyldeoxygalactonojirimycin in which the alkyl isbutyl.